Nodal signaling is required for closure of the anterior neural tube in zebrafish.

Aquilina-Beck A, Ilagan K, Liu Q, Liang JO - BMC Dev. Biol. (2007)

Bottom Line:
N-cadherin expression and localization to the membrane are reduced in fish that lack Nodal signaling.Overexpression of an activated form of the TGFbeta Type I receptor Taram-A (Taram-A*) cell autonomously rescues mesendoderm formation in fish with a severe decrease in Nodal signaling.This work helps establish a role for Nodal signals in neurulation, and suggests that defects in Nodal signaling could underlie human neural tube defects such as exencephaly, a fatal condition characterized by an open neural tube in the anterior brain.

Background: Nodals are secreted signaling proteins with many roles in vertebrate development. Here, we identify a new role for Nodal signaling in regulating closure of the rostral neural tube of zebrafish.

Results: We find that the neural tube in the presumptive forebrain fails to close in zebrafish Nodal signaling mutants. For instance, the cells that will give rise to the pineal organ fail to move from the lateral edges of the neural plate to the midline of the diencephalon. The open neural tube in Nodal signaling mutants may be due in part to reduced function of N-cadherin, a cell adhesion molecule expressed in the neural tube and required for neural tube closure. N-cadherin expression and localization to the membrane are reduced in fish that lack Nodal signaling. Further, N-cadherin mutants and morphants have a pineal phenotype similar to that of mutants with deficiencies in the Nodal pathway. Overexpression of an activated form of the TGFbeta Type I receptor Taram-A (Taram-A*) cell autonomously rescues mesendoderm formation in fish with a severe decrease in Nodal signaling. We find that overexpression of Taram-A* also corrects their open neural tube defect. This suggests that, as in mammals, the mesoderm and endoderm have an important role in regulating closure of the anterior neural tube of zebrafish.

Conclusion: This work helps establish a role for Nodal signals in neurulation, and suggests that defects in Nodal signaling could underlie human neural tube defects such as exencephaly, a fatal condition characterized by an open neural tube in the anterior brain.

Figure 2: Elongated and divided phenotypes in squint mutants persist through the first three days of development. The morphology of the pineal was followed over time in individual sqt mutants and WT siblings carrying the flh:eGFP transgene, which expresses GFP throughout the pineal anlage during embryogenesis [21]. All sqt mutants used in this experiment had cyclopic eyes. (A-D) Composite bright field and fluorescent images at 2 dpf showing fluorescence in the pineal anlage of embryos having (A-B) normal, round-shaped pineal anlagen, and (C) elongated and (D) divided pineal anlagen. (E-H") Each row shows the pineal of an individual embryo over three days of development as assayed by fluorescence microscopy on a compound microscope. Note the similarity in the morphology of the pineal in (E-E") the WT embryo and (F-F") one of the sqt mutants. In contrast, the sqt mutants in G-G" and H-H" maintain their abnormal elongated and divided morphologies throughout the experiment. All images are dorsal views with anterior to top. Scale bars: 60 μm (A-D), 30 μm (E-H").

Mentions:
To characterize the phenotype of sqt mutants more fully, morphology of the developing pineal was followed over time in individual WT and sqt embryos carrying the flh:eGFP transgene, which drives eGFP expression in the pineal [21]. Homozygous sqt mutants were unambiguously identified by their cyclopic eye phenotype. Siblings with two normal eyes could have had one of three genotypes; +/+, sqt/+, or sqt/sqt. Fish with normal, elongated, and divided pineal phenotypes were identified by fluorescence microscopy at 1 dpf and then followed for the next two days (Figure 2). In sqt mutants with a round shaped pineal at 1 dpf (Figure 2B, and 2F–F"), the pineal anlage remained indistinguishable from their WT siblings through the following two days (Figure 2E–F"). In sqt mutants with an elongated or divided pineal at 1 dpf (Figure 2C, D, and 2G–H"), the abnormal pineal morphology persisted or became even more severe over time (Figure 2G–H"). If the elongated/divided pineal phenotypes were merely due to a delay in development, then they should have been rectified over the three days of the experiment. The persistence of these phenotypes suggests that they are instead due to a defect in the mechanism that drives convergence of the pineal precursors to the midline of the diencephalon.

Figure 2: Elongated and divided phenotypes in squint mutants persist through the first three days of development. The morphology of the pineal was followed over time in individual sqt mutants and WT siblings carrying the flh:eGFP transgene, which expresses GFP throughout the pineal anlage during embryogenesis [21]. All sqt mutants used in this experiment had cyclopic eyes. (A-D) Composite bright field and fluorescent images at 2 dpf showing fluorescence in the pineal anlage of embryos having (A-B) normal, round-shaped pineal anlagen, and (C) elongated and (D) divided pineal anlagen. (E-H") Each row shows the pineal of an individual embryo over three days of development as assayed by fluorescence microscopy on a compound microscope. Note the similarity in the morphology of the pineal in (E-E") the WT embryo and (F-F") one of the sqt mutants. In contrast, the sqt mutants in G-G" and H-H" maintain their abnormal elongated and divided morphologies throughout the experiment. All images are dorsal views with anterior to top. Scale bars: 60 μm (A-D), 30 μm (E-H").

Mentions:
To characterize the phenotype of sqt mutants more fully, morphology of the developing pineal was followed over time in individual WT and sqt embryos carrying the flh:eGFP transgene, which drives eGFP expression in the pineal [21]. Homozygous sqt mutants were unambiguously identified by their cyclopic eye phenotype. Siblings with two normal eyes could have had one of three genotypes; +/+, sqt/+, or sqt/sqt. Fish with normal, elongated, and divided pineal phenotypes were identified by fluorescence microscopy at 1 dpf and then followed for the next two days (Figure 2). In sqt mutants with a round shaped pineal at 1 dpf (Figure 2B, and 2F–F"), the pineal anlage remained indistinguishable from their WT siblings through the following two days (Figure 2E–F"). In sqt mutants with an elongated or divided pineal at 1 dpf (Figure 2C, D, and 2G–H"), the abnormal pineal morphology persisted or became even more severe over time (Figure 2G–H"). If the elongated/divided pineal phenotypes were merely due to a delay in development, then they should have been rectified over the three days of the experiment. The persistence of these phenotypes suggests that they are instead due to a defect in the mechanism that drives convergence of the pineal precursors to the midline of the diencephalon.

Bottom Line:
N-cadherin expression and localization to the membrane are reduced in fish that lack Nodal signaling.Overexpression of an activated form of the TGFbeta Type I receptor Taram-A (Taram-A*) cell autonomously rescues mesendoderm formation in fish with a severe decrease in Nodal signaling.This work helps establish a role for Nodal signals in neurulation, and suggests that defects in Nodal signaling could underlie human neural tube defects such as exencephaly, a fatal condition characterized by an open neural tube in the anterior brain.

Background: Nodals are secreted signaling proteins with many roles in vertebrate development. Here, we identify a new role for Nodal signaling in regulating closure of the rostral neural tube of zebrafish.

Results: We find that the neural tube in the presumptive forebrain fails to close in zebrafish Nodal signaling mutants. For instance, the cells that will give rise to the pineal organ fail to move from the lateral edges of the neural plate to the midline of the diencephalon. The open neural tube in Nodal signaling mutants may be due in part to reduced function of N-cadherin, a cell adhesion molecule expressed in the neural tube and required for neural tube closure. N-cadherin expression and localization to the membrane are reduced in fish that lack Nodal signaling. Further, N-cadherin mutants and morphants have a pineal phenotype similar to that of mutants with deficiencies in the Nodal pathway. Overexpression of an activated form of the TGFbeta Type I receptor Taram-A (Taram-A*) cell autonomously rescues mesendoderm formation in fish with a severe decrease in Nodal signaling. We find that overexpression of Taram-A* also corrects their open neural tube defect. This suggests that, as in mammals, the mesoderm and endoderm have an important role in regulating closure of the anterior neural tube of zebrafish.

Conclusion: This work helps establish a role for Nodal signals in neurulation, and suggests that defects in Nodal signaling could underlie human neural tube defects such as exencephaly, a fatal condition characterized by an open neural tube in the anterior brain.